Proof of the Mysterious Efficacy of Ginseng: Basic and Clinical Trials

Journal of Pharmacological Sciences
J Pharmacol Sci 95, 000 – 000 (2004)
©2004 The Japanese Pharmacological Society
Forum Minireview
Proof of the Mysterious Efficacy of Ginseng: Basic and Clinical Trials:
Metabolic Activation of Ginsenoside: Deglycosylation by Intestinal
Bacteria and Esterification with Fatty Acid
Hideo Hasegawa1,*
1
Fermenta Herb Institute Inc., 1-30-15 Motohongo, Hachioji, Tokyo 192-0051, Japan
Received February 12, 2004; Accepted March 26, 2004
Abstract. Orally ingested ginsenoside passes through the stomach and small intestine without
decomposition by either gastric juice or liver enzymes into the large intestine, where ginsenoside
is deglycosylated by colonic bacteria followed by transit to the circulation. Colonic bacteria
cleave the oligosaccharide connected to the aglycone stepwise from the terminal sugar to afford
the major metabolites, 20S-protopanaxadiol 20-O-b-D-glucopyranoside (M1) and 20S-protopanaxatriol (M4). These metabolites are further esterified with fatty acids. The resultant fattyacid conjugates are still active molecules that are sustained longer in the body than parental
metabolites. Accumulating evidence strongly suggests that ginsenoside is a prodrug that is
activated in the body by intestinal bacterial deglycosylation and fatty acid esterification.
Keywords: ginsenoside, metabolic activation, intestinal bacteria, deglycosylation,
fatty acid esterification
contributed evidence to their hypothesis and also
expanded the research field of plant glycosides (8). This
review describes the metabolic activation of ginsenoside
in the body.
Introduction
In the latter of the 20th century, the pharmaceutical
studies on ginseng have started in parallel with structural
elucidation of its ingredients, in order to give scientific
grounds to the medical efficacy of ginseng. So far,
numerous researchers have contributed to the accumulation of evidence that ginsenoside is responsible for the
pharmacological effects of ginseng. Indeed, ginsenoside
itself exerted various pharmacological activities, by
directly being added to cell cultures in vitro or by being
intraperitoneally or intravenously injected to experimental animals. These results led to the misunderstanding that intact ginsenoside might be the real active
principle in the body. However, Kobashi et al. have
proposed the concept that plant glycoside acts as a
prodrug that is metabolized to the active form by
intestinal bacterial deglycosylation (1, 2). We have
revealed that the anticancer activities of ginsenoside
after oral administration are based on its metabolites
formed by intestinal bacterial deglycosylation (3, 4)
and fatty-acid esterification (5 – 7). These data have
Deglycosylation of ginsenoside by intestinal bacteria
Ginseng is orally ingested, in general. Therefore, its
ingredients must meet gastric juice, digestive and
bacterial enzymes in the intestines. Ginsenoside is
hardly decomposed by gastric juice with the exception
of slight oxygenation (9); however, the oral bioavailabilities of intact ginsenosides from the intestines are
extremely low (Rb1, 0.1% to 4.4%; Rb2, 3.7%; Rg1, 1.9%
to 18.4%) (10 – 12). In spite of poor absorption of
ginsenoside, radioisotope assay revealed that serum
radioactivity in rats orally given Rb2 together with its
radioactive derivative was 3 times higher than the serum
Rb2-level determined by HPLC (11).
Our results from incubation of Rb1 with the contents
of mouse gastrointestinal tract showed that ginsenoside
is metabolized to 20S-protopanaxadiol 20-O-b-Dglucopyranoside (M1, Table 1) only by the contents of
the large intestine (13). This phenomenon was also
observed in the mice orally administered with Rb1. With
*Corresponding author. FAX: +81-426-24-1734
E-mail: [email protected]
1
2
H Hasegawa
Table 1.
Chemical structures of ginsenosides and their metabolites formed by intestinal bacteria
Ginsenoside
R1
R2
R3
O-Glc2-1Glc
O-Glc2-1Glc
O-Glc2-1Glc
O-Glc2-1Glc
H
H
H
H
O-Glc6-1Glc
O-Glc6-1Arap
O-Glc6-1Araf
O-Glc
OH
OH
O-Glc2-1Rha
O-Glc
O-Glc
O-Glc
O-Glc2-1Glc
O-Glc
O-Glc
O-Glc
OH
O-Glc
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
O-Glc
O-Glc6-1Glc
O-Glc6-1Araf
O-Glc6-1Arap
O-Glc6-1Glc
O-Glc
O-Glc6-1Araf
O-Glc6-1Arap
O-Glc
OH
OH
OH
OH
O-Glc
OH
OH
OH
O-Glc
OH
Major ginsenoside
20S-Protopanaxadiol type:
Rb1
Rb2
Rc
Rd
20S-Protopanaxatriol type:
Re
Rg1
Intestinal bacterial metabolite
20S-Protopanaxadiol type:
M10 (Rd)
M9 (Gp-XVII)
M7 (Mb)
M6
M13 (Gp-LXXV)
M5 (F2)
M3 (Mc)
M2 (C-Y)
M1 (C-K)
M12 (aglycone)
20S-Protopanaxatriol type:
M8 (Rh1)
M11 (F1)
M4 (aglycone)
Glc, b -D-glucopyranose; Arap, a-L-arabinopyranose; Araf, a-L-arabinofuranose; Rha, a-L-rhamnopyranose; Gp,
gypenoside; C-K, compound K; C-Y, compound Y.
the passage of time after administration, Rb1 was
detected in the stomach and small intestine followed by
M1 in the caecum and colorectum (13). These results
indicate that intestinal bacteria metabolize Rb1 to M1.
Akao et al. detected M1, which they called compound K,
in the plasma of gnotobiotic rats orally administered
with Rb1 (14). Recently, Tawab et al. provided evidence
that M1, M8, and M11 may reach the circulation in
humans after ginseng ingestion (15).
Intestinal bacteria cleave the oligosaccharides connected to the C-3 or C-20 hydroxyl group of the
aglycone stepwise from the terminal sugar (16, 17).
The main metabolic pathways are supposed to be as
follows: protopanaxadiol-type, Rb1®[M10 (Rd)®M5
(F2) or M9®M13]®M1, Rb2®M6®M2®M1, and
Rc®M7®M3®M1 (M1 is gradually hydrolyzed to
M12); protopanaxatriol-type, Re®Rg1®M11 (F1) or
M8 (Rh1)®M4 (Table 1). Many kinds of bacteria
including Prevotella oris (18), Eubacterium A-44 (14),
Bifidobacterium K506 (17), Bacteroides JY6 (17),
and Fusobacterium K-60 (17) seem to cooperatively
metabolize ginsenoside.
Metabolic Activation of Ginsenoside
3
Fig. 1. Putative metabolic pathways of ginsenosides in the body after oral administration. Ginsenosides are deglycosylated
to M1 or M4 by intestinal bacteria followed by absorption into the blood or the mesenteric lymphatics. Although M1 is mostly
excreted as bile, some M1 may be esterified with fatty acids at C-3 of the aglycone moiety or C'-6 of the glucose moiety in
the liver. EM1 is not excreted in the small intestine and so it is accumulated in the liver longer than M1. On the contrary, most
M4 is esterified with fatty acids and accumulated in the tissues including the liver and lung followed by excretion of esterified
M4 (EM4) as bile.
Esterification of intestinal bacterial metabolite with
fatty acid
Following intravenous administration of M1 to rats
and mice, it rapidly disappeared from the circulation
(t1 / 2a , 3 min; t1/ 2b , 23 min; AUC, 2815 min × mg / ml) and
instead, increased in the liver. The hepatic M1-level
peaked at 10 min (Cmax, 65% recovery) and thereafter
gradually disappeared with time (t1/ 2a , 25 min; t1/ 2b ,
75 min; AUC, 481.4 h × m g/ g). Although most M1 was
excreted as bile, approximately 24 mol% of dosed M1
was esterified with fatty acids in the liver without
structural variation. Esterified M1 (EM1) was not
excreted in the small intestine and so EM1 was accumulated in the liver longer than M1, thus suggesting that
metabolic regulation differs between M1 and EM1.
Mass spectrometry analysis defined EM1 as a mixture of
various fatty acid M1 mono-esters, including stearate,
oleate, or palmitate (5). The fragment pattern of EM1
suggests that fatty acid is linked to M1 at C-3 of the
aglycone moiety or C'-6 of the glucose moiety.
In the case of M4 (7), orally ingested M4 was
4
H Hasegawa
completely absorbed from the small intestine into the
mesenteric lymphatics. Then rapid fatty acid esterification of M4 occurred at C-3 of the aglycone moiety.
Esterified M4 (EM4) spread to other organs in the
body followed by its excretion as bile. Inconsistent with
M1, the selective accumulation of M4 in the liver after
its intravenous administration was not observed. The
structural difference between M1 and M4 is the glucose
moiety connected at C-20 of the aglycone (Table 1).
Hepatocytes are shown to recognize glucose moiety via
a receptor (19, 20). This specific function of hepatocytes
must be partly associated with the selective accumulation of M1 into the liver. Based on these results, putative
metabolic pathways of ginsenoside after oral administration are proposed in Fig. 1.
To our knowledge, there have been few reports on
animal fatty acid triterpene esters, except for 2 reports by
Tabas et al. who isolated the triterpene esters containing
stearate and palmitate from the liver of rabbits and
humans (21). They hypothesized that the origin of fatty
acid triterpene esters may be via dietary absorption of
plant triterpenes followed by fatty acid esterification of
Table 2.
the triterpene in animal tissues (22). The detection of
EM1 and EM4 in the tissues is the first evidence for
their hypothesis.
Metabolic regulation of ginsenoside metabolites (M1
and M4) differed from those glycosides which are
conjugated with glucronic acid (ex., glycyrrhetic acid
and baicalein, the sapogenins of glycyrrhizin and
baicalin) (1).
Pharmacological activity of ginsenoside metabolite
An issue raised here is whether ginsenoside metabolites are active or not. The results from the in vivo and
in vitro comparative examinations using ginsenoside
and its intestinal bacterial metabolite are as follows
(2, 3): When administered orally (ginsenoside is hydrolyzed to metabolite by intestinal bacteria), both ginsenoside and metabolite were effective in inhibiting tumor
metastasis. However, the activity of orally ingested
ginsenoside was correlated with the ginsenoside-hydrolyzing potential of intestinal bacteria (23). In the case of
intravenous administration (metabolite is not produced
Pharmacological activity of ginsenoside metabolites cited in literature
Ginsenoside metabolite
assay condition
pharmacological potential (Reference No.)
Intestinal bacterial metabolite
20(S)-Protopanaxadiol type:
M3 (Mc)
M1 (C-K)
in vitro
in vitro
in vivo
M12 (aglycone)
20(S)-Protopanaxatriol type:
M4 (aglycone)
in vitro
in vitro
in vivo
anti-inflammatory activity (17)
inhibitor of tumor cell proliferation (2, 27)
inhibitor of tumor-induced neovascularization (25)
inducer of apoptotic cell death (26)
regulator of tumor cell cycle (27)
anti-inflammatory activity (17)
inhibitor of tumor growth (25)
inhibitor of tumor metastasis (2, 23)
inhibitor of carcinogenesis (13)
regulator of hypothalamo-pituitary-adrenal axis (28)
inhibitor of Helicobacter pylori (29)
inhibitor of tumor growth (17)
inhibitor of catecholamine secretion (30)
inhibitor of tumor cell proliferation (3)
inhibitor of tumor metastasis (3)
Fatty acid ester of intestinal bacterial metabolite
20(S)-Protopanaxadiol type:
Fatty acid M1 ester (EM1)
20(S)-Protopanaxatriol type:
Fatty acid M4 ester (EM4)
in vitro
in vivo
immunomodulator (6)
inhibitor of tumor growth (5, 6)
inhibitor of tumor metastasis (5, 6)
in vitro
potentiator of natural killer activity (7)
Metabolic Activation of Ginsenoside
from ginsenoside), not ginsenoside but metabolite was
effective (2, 3). Tumor invasion into extracellular
matrices and basement membranes is a crucial step in the
complex multistage process that leads to the metastatic
formation. Under in vitro conditions, slight or no inhibition of tumor invasion was seen using ginsenoside,
whereas the metabolite even at non-cytotoxic concentrations inhibited tumor invasion more effectively than
ginsenoside (2, 3). These findings suggest that the
action of bacterial metabolites primarily mediates the
antimetastatic effects by orally ingested ginsenoside.
This concept agrees with the pharmacokinetic findings:
not ginsenosides but their bacterial metabolites are
absorbed from the intestines after oral administration of
ginsenosides.
The in vitro cytotoxicity of metabolites was
attenuated by fatty acid esterification (6), implying that
the esterification is a detoxification reaction, just as
cholesterol esterification is shown to prevent the cytotoxicity of excess intracellular cholesterol (24). Metabolites did not directly affect tumor growth in vitro (6),
whereas they stimulated lymphocytes to become cytotoxic to tumor cells (6, 7). Concerning the pharmacokinetics, both M1 and EM1 were selectively taken up
into the liver soon after intravenous administration.
Thereafter, M1 was cleared immediately from the liver;
however, EM1 was retained in the liver at a level of
more than 25% of the administered dose for 24 h
after administration (6). These findings suggest that
fatty acid esterification of intestinal bacterial metabolite
of ginsenoside potentiates the antitumor activity of the
parental metabolites through delay of the clearance and
through immunostimulation.
The pharmacological activities of ginsenoside metabolites are summarized in Table 2.
Conclusion
Orally ingested ginsenoside is activated by intestinal
bacterial deglycosylation followed by fatty acid esterification. Therefore, the deglycosylation process of ginsenoside is crucial for its pharmacological expression.
Intestinal bacteria, however, are very changeable in
dependence of host conditions, including diet, health,
and even stress. Indeed, bacterial ginsenoside-hydrolyzing potentials have been shown to differ among humans
(18) and experimental mice (13). Therefore, it is easily
hypothesized that the individual differences in bacterial
ginsenoside-hydrolyzing potentials may affect ginseng
efficacy. Fermented ginseng products containing ginsenoside metabolites may have merit for standardizing
ginseng efficacy.
5
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